Estradiol (1,3,5(10)-Estratrien-3,17a-diol) is secreted by the ovary and placenta. It is synthesized by the aromatization of androgens in the thecal and granulosa cells of the ovary and placenta. The aromatization is stimulated by follitropin (FSH). Estradiol synthesis in turn stimulates production of Lutropin (LH) receptors necessary for the synthesis of androgen precursors.
Estradiol is important for female sexual differentiation during gestation, sexual development at the onset of puberty, and regulation of the menstrual cycle. The menstrual cycle is the result of a precise coordination of the functional characteristics of the Central Nervous System, the hypothalamus, the pituitary, the ovary, and the endometrium which regulate the cyclic release of Gonadotropin Releasing Hormone (GnRH), LH and FSH, and ovarian steroids (Estradiol and Progesterone). Estradiol is involved in both the stimulation and inhibition of the release of the gonadotropins, exerting both a positive and a negative feedback. Early in the follicular phase, ovarian secretion of estradiol from the thecal and granulosa cells is modest. During the follicular phase, estradiol stimulates endometrial growth (repairing the endometrium after menses). Toward mid-cycle, LH production increases and results in the release of the ovum by the rupture of the developed follicle. After ovulation, estradiol secretion declines slightly. During the luteal phase, estradiol along with progesterone are secreted by the corpus luteum stimulating further endometrial growth. If the ovum is not fertilized, there is a further drop in estradiol and progesterone. This drop in estradiol and progesterone initiates menses.
The measurement of estradiol is important for the evaluation of normal sexual development (menarche), causes of infertility (anovulation, amenorrhea, dysmenorrhea), and menopause. Normal estradiol levels are lowest at menses and during the early follicular phase (25-75 pg/ml). The levels rise in the late follicular phase to a peak of 200-600 pg/mL just before the LH surge initiates ovulation. As LH peaks, estradiol begins to decrease before rising again during the luteal phase (100-300 pg/mL). If conception does not take place, estradiol falls further to its lowest levels, thus initiating menses. If conception occurs, estradiol levels continue to rise, reaching levels of 1-5 ng/ml during the first trimester, 5-15 ng/ml during the second trimester, and 10-40 ng/ml during the third trimester. During menopause, estradiol levels remain low.
There are various methods for measuring estradial levels in serum. However, each of these methods utilize radioactive elements as labels and suffer from several disadvantages. The methods require several different steps including manual steps, several vessels, and are either nonautomated or only semiautomated. Radiolabels are problematic because the expense involved in such assays are much greater than in non-radiolabeled assay formats. For example, more highly skilled laboratory personnel are required and waste disposal is more tightly controlled and regulated. The following are commercially available radioimmunoassay methods for determining estradiol level in serum samples.
One assay for estradiol (commercially available from Pantex) is a direct, non-extraction competitive assay. Radiolabeled estradiol tracer, anti-estradiol antibody and sample are mixed and incubated for two hours. Anti-antibody antibody is added and the mixture is incubated for 15 minutes to form a precipitate. The mixture is centrifuged to pellet the precipitate formed. The supernatant is decanted and the radioactivity of the precipitant is measured. The radioactivity measured is compared to a radioactivity vs. estradiol concentration plot to determine the estradiol concentration in the sample. The following compounds are known to cross-react in the assay: .alpha.-Estradiol (1.4%) and Danazol (0.6%).
Another assay for estradiol (Coat-A-Count Radioimmunoassay (RIA) for Estradiol commercially available from Diagnostic Products Corporation) is a direct, non-extraction antibody coated tube competitive assay. Each tube is coated with anti-estradiol antibody. Radiolabeled estradiol tracer and sample are incubated in the tube for three (3) hours. After decanting the mixture and washing the tube, the radioactivity of the tube is measured. The radioactivity measured is compared to a radioactivity vs. estradiol concentration plot to determine the estradiol concentration in the sample. The following compounds are known to cross-react in the assay: Ethinyl Estradiol (1.8%); Estrone (1.1%); Estradiol-3.beta.-D-glucuronide (0.7%); Estradiol-3-Sulfate (0.3%); and 19-Nortestosterone (0.25%).
Yet another assay for estradiol (Estradiol MAIA commercially available from Serono)is also a direct, non-extraction competitive assay. Radiolabeled estradiol tracer, anti-estradiol antibody and the sample are incubated for one to three hours (depending on the assay sensitivity and precision desired) to permit the formation of antibody-estradiol complexes. The complexes are separated from the sample by incubation of the reaction mixture with anti-antibody antibody coated magnetic particles followed by sedimentation of the magnetic particles through the application of a magnetic field. The separated particles are then washed. The measured radioactivity level of the particles is compared to a radioactivity vs. estradiol concentration plot to determine the estradiol concentration in the sample. The following compounds are known to cross-react in the assay: Estrone (2.6%); EstradioI-Dipropionate (0.3%); Estradiol-3.beta.-D-glucuronide (0.2%); and Estriol (0.2%).
Some non-radiolabeled immunoassays have been reported, but do not have the sensitivity or specificity needed to accurately and precisely measure estradiol levels. Dawson et al. (Steroids, 31:357-366, 1978) tested rabbit polyclonal anti-estradiol antibodies using horseradish peroxidase coupled to estrone at carbon 11. The cross-reactivities of the antibodies to estrone ranged from 5% to 120%. Such cross-reactivities would not be acceptable in an estradiol specific assay. Thus, an enzyme immunoassay that utilizes a conjugate where the enzyme is coupled to position 11 of estrone would not likely produce a satisfactory assay.
Pandey et al. (Clinica Chimica Acta, 190:175-184, 1990) reported an enzyme-linked immunosorbant assay (ELISA) using the conjugate estradiol-6-(O-carboxymethyl)oxime linked to penicillinase with anti-estradiol antibody coated wells of a microtiter plate. The assay had a lower sensitivity limit of 25 pg/mL, but required a two (2) hour incubation of the reaction mixture at 37.degree. C. and after washing, the enzyme substrate/enzyme reaction required incubation at 37.degree. C. for one (1) hour. Thus the overall assay time was in excess of three (3) hours. Maurel et al. (J. Immunolog. Methods, 102:165-172, 1987) also reported an ELISA using an estradiol-6-(O-carboxymethyl)oxime conjugated to .beta.-galactosidase, which had improved sensitivity over Pandey et al.'s assay. However, as in Pandey et al.'s method, Maurel et al.'s method requires extensive incubation times. The anti-estradiol antibody coated on microtiter wells are incubated with the sample for ninety (90) minutes at 37.degree. C., then incubated with conjugate for ninety (90) minutes, and then after washing the enzyme activity was determined after two (2 ) hours at 42.degree. C. Thus, the assay time was well in excess of three (3) hours.
De Boever et al. (Clin. Chem., 32:1895-1900, 1986) reported a chemiluminescence immunoassay for estradiol with a sensitivity limit of about 49 pg/mL and an assay time of in excess of ninety (90) minutes. Roda et al. (Anal. Blochem., 156:267-273, 1986) reported a luminescent enzyme immunoassay for estradiol which required over four (4) hours to perform.
De Lauzon et al. (J. Immunoassay, 10:339-357, 1989) reported a competitive enzyme immunoassay for estradiol using microtiter plate wells coated with estradiol coupled to bovine serum albumin (BSA). The sample and peroxidase labeled anti-estradiol antibody were incubated in the wells for two (2) hours at room temperature. Alternatively, biotinylated anti-estradiol antibodies were utilized folilowed by a second incubation of three (3) hours with avidin coupled to peroxidase.
Clearly, there is a need for an estradiol non-radiolabel immunoassay that is rapid, accurate, sensitive, easy to perform, free from interferences and relatively insensitive to experimental variables such as pH and temperature. An object of the present invention is to develop an assay method and reagents to perform estradiol measurements accurately and with precision without the need of a radiolabeled tracer. Because of the very low concentrations of estradiol present in biological fluid samples (0.025-40 ng/mL), any alternative method must be very sensitive. The existing methods utilize long incubation times to overcome this sensitivity requirement. Another object of this invention is to eliminate the long incubation times required in non-radiolabeled immunoassays.